Post-synthetic chemical modification of rna at the 2&#39;-position of the ribose ring via &#34;click&#34; chemistry

ABSTRACT

This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128). The invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved cellularmechanism of post-transcriptional gene silencing found in fungi, plantsand animals that uses small RNA molecules to inhibit gene expression ina sequence-specific manner. The RNAi machinery can be harnessed todestruct any mRNA of a known sequence. This allows for suppression(knock-down) of any gene from which it was generated and consequentlypreventing the synthesis of the target protein. Smaller siRNA duplexesintroduced exogenously were found to be equally effective triggers ofRNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Bartel, D. P. Cell 2000,101, 25-33). Synthetic RNA duplexes can be used to modulatetherapeutically relevant biochemical pathways, including ones which arenot accessible through traditional small molecule control.

Chemical modification of RNA leads to improved physical and biologicalproperties such as nuclease stability (Damha et al Drug Discovery Today2008, 13(19/20), 842-855), reduced immune stimulation (Sioud TRENDS inMolecular Medicine 2006, 12(4), 167-176), enhanced binding (Koller, E.et al Nucl. Acids Res. 2006, 34, 4467-4476), enhanced lipophiliccharacter to improve cellular uptake and delivery to the cytoplasm.

Chemical modifications of RNA have relied heavily on work-intensive,cumbersome, multi-step syntheses of structurally novel nucleosideanalogues and their corresponding phosphoramidites prior to RNAassembly. In particular, a major emphasis has been placed on chemicalmodification of the 2′-position of nucleosides. A rigorous approach tostructure-activity-relationship (SAR) studies of chemical modificationswill obviously require synthesis and evaluation of all four canonicalribonucleosides [adenosine (A), cytidine (C), uridine (U), guanosine(G)]. Furthermore, some chemical modifications bear sensitive functionalgroups that may be incompatible with state-of-the-art automatedsynthesis of RNA as well as subsequent downstream cleavage-deprotectionsteps. These attributes have made chemical modification of RNA prior tosynthesis rather low-throughput and limited in scope.

Post-synthetic chemical modifications of RNA have centered for the mostpart on simple conjugation chemistry. Conjugation has largely beenperformed on either the 3′ or the 5′-end of the RNA via alkylamine anddisulfide linkers. These modifications have allowed conjugation of RNAto various compounds such as cholesterol, fatty acids,poly(ethylene)glycols, various delivery vehicles and targeting agentssuch as poly(amines), peptides, peptidomimetics, and carbohydrates.

This invention relates to the post-synthetic chemical modification ofRNA at the 2′-position on the ribose ring via a copper catalyzed Huisgencycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today2003, 8, 1128). The invention 1) avoids complex, tedious multi-stepsyntheses of each desired modified ribonucleoside; 2) allows diversechemical modifications using high-fidelity chemistry that is completelyorthogonal to commonly used alkylamino, carboxylate and disulfide linkerreactivities; 3) allows introduction of functional groups that areincompatible with modern automated solid-phase synthesis of RNA andsubsequent cleavage-deprotection steps; 4) allows introduction offunctional groups useful as targeting ligands; and 5) enableshigh-throughput structure-activity relationship studies on chemicallymodified RNA in 96-well format.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Systematic evaluation of the impact on knockdown of the2′-O-benzyl-triazole inosine chemical modification along positions 1through 19 of the guide strand of a SSB(291) siRNA.

FIG. 2. Systematic evaluation of the impact on knockdown of the2′-O-phenylthiomethyl-triazole inosine chemical modification alongpositions 1 through 19 of the guide strand of a SSB(291) siRNA.

FIG. 3. Systematic evaluation of the impact on knockdown of the2′-O-benzyl-triazole inosine chemical modification was systematicallyevaluated along positions 1 through 19 of the guide strand of a Luc(80)siRNA.

FIG. 4. Systematic evaluation of the impact on knockdown of the2′-O-phenylthiomethyl-triazole inosine chemical modification wassystematically evaluated along positions 1 through 19 of the guidestrand of a Luc(80) siRNA.

FIG. 5. Duration of knockdown activity of the 2′-O-benzyl-triazoleinosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of a Luc(80) siRNA.

FIG. 6. Duration of knockdown activity of the 2′-O-phenylthiornethylinosine chemical modification was systematically evaluated alongpositions 1 through 19 of the guide strand of a Luc(80) siRNA.

FIG. 7. Introduction of N-acetyl-galactosamine as chemical modification.

FIG. 8. Introduction of poly(ethylene)glycol amine in SSB(291) RNA.

FIG. 9. Multi-click for introduction of multiple N-acetylgalactosaminechemical modifications in one synthetic operation.

SUMMARY OF THE INVENTION

This invention relates to the post-synthetic chemical modification ofRNA at the 2′-position on the ribose ring via a copper catalyzed Huisgencycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today2003, 8, 1128). The invention 1) avoids complex, tedious multi-stepsyntheses of each desired modified ribonucleoside; 2) allows diversechemical modifications using high-fidelity chemistry that is completelyorthogonal to commonly used alkylamino, carboxylate and disulfide linkerreactivities; 3) allows introduction of functional groups that areincompatible with modern automated solid-phase synthesis of RNA andsubsequent cleavage-deprotection steps; 4) allows introduction offunctional groups useful as targeting ligands; and 5) enableshigh-throughput structure-activity relationship studies on chemicallymodified RNA in 96-well format.

DETAILED DESCRIPTION OF THE INVENTION

Methods for the synthesis of nucleotide derivatives wherein molecules ofinterest are grafted on the oligonucleotide with the help of a “clickchemistry” reaction between an azide function on the molecule ofinterest and an alkyne function on the oligonucleotide are demonstratedin US 2009/0124571. This work discloses molecules such as carbohydrates,peptides, lipids, oligonucleotides, biotin, ferrocenyl compounds andfluorescent tags which are grafted onto oligonucleotides utilizingalkyne phosphoester derivatives of the oligonucleotides to make the1,3-cycloaddition with an azide-substituted molecule of interest.

Methods for forming azido-modified nucleic acid conjugates of reportermolecules, carrier molecules or solid support utilizing “clickchemistry” are disclosed in US 2008/0050731.

Synthesis of modified RNA and DNA utilizing an alkyne handle on a baseand subsequent “click chemistry” is disclosed in WO 2008/052775 and inCN 101550175.

Recent reviews regarding “click chemistry” and oligonucleotide synthesisare covered by Gramlich et al. Angew. Chem. Int. Ed. 2008, 47,8350-8358; Amblard et al. Chem.

Rev. 2009, 109, 4207-4220.

Thus the prior art discloses the use of “click chemistry” to generatemodified oligonucleotides wherein the alkyne functional group is on thephosphate backbone or the base in DNA and RNA molecules or the alkynefunctional group is on the ribose of DNA molecules. Typically, themodification is for labeling purposes.

The use of “click chemistry” to generate 2′-modified RNA wherein thealkyne functional group is on the ribose is not known. There areconsiderable differences in the physico-chemical properties of RNA andDNA. For example, it is well recognized that RNA is much less stablethan DNA towards hydrolysis. Furthermore, RNA can undergo auto-catalyticcleavage via intramolecular cyclization of the 2′-position onto the3′-phosphodiester. Modification of the 2′-position is critical for RNAstability and therapeutic applicability. RNA with alkyne functionalgroup at the 2′-position.

The current invention relates to chemical modification of RNA at the2′-position of the ribose ring based on the 1,3-dipolar cycloaddition(Huisgen reaction) between alkynes and azides. The 1,3-dipolarcycloaddition (Huisgen reaction) between alkynes and azides is known.(Tornøe, Christensen, Meldal J. Org. Chem. 2002, 67, 3057; Rostovstev,Green, Fokin, Sharpless Angew. Chem. Int. Ed. 2002, 41, 2596).

In an embodiment, the invention provides a process for introducing2′-modifications into RNA, said process comprises a) obtaining RNA withan alkyne functional group at the 2′-position on at least one ribosering; b) creating a solution of RNA in a solvent; and c) adding anorganic azide and a metal catalyst to the solution to form a reactionand creating a 2′-modified RNA.

In an embodiment, the process is conducted in high-throughput format.

In an embodiment, the step (a) RNA may be purchased or synthesized.

In an embodiment, the step (b) solvent is selected from aqueous buffersolutions (including phosphate buffers), aqueous DMSO, CH₃CN, DMF, DMAc,NMP and a suitable ionic liquid.

In an embodiment, the step (b) solvent is aqueous DMSO.

In an embodiment, the step (c) metal catalyst is selected from copperand ruthenium.

In an embodiment, the step (c) metal catalyst is copper.

In an embodiment, the step (c) metal catalyst is copper with a suitableligand to stabilize the Cu(I) oxidation state.

In an embodiment, the step (c) reaction is performed at temperaturesbetween −20-300° C. for 0 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 5-120° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 20-100° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 60-90° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 65-80° C. for 0.5 to 18 h.

In another embodiment, the invention provides a process for introducing2′-modifications into RNA, said process comprises a) obtaining RNA withan alkyne functional group at the 2′-position on at least one ribosering of an internal nucleotide; b) creating a solution of RNA in asolvent; and c) adding an organic azide and a metal catalyst to thesolution to form a reaction and creating a 2′-modified RNA.

In an embodiment, the process is conducted in high-throughput format.

In an embodiment, the step (a) RNA may be purchased or synthesized.

In an embodiment, the step (b) solvent is selected from aqueous buffersolutions (including phosphate buffers), aqueous DMSO, CH₃CN, DMF, DMAc,NMP and a suitable ionic liquid.

In an embodiment, the step (b) solvent is aqueous DMSO.

In an embodiment, the step (c) metal catalyst is selected from copperand ruthenium.

In an embodiment, the step (c) metal catalyst is copper.

In an embodiment, the step (c) metal catalyst is copper with a suitableligand to stabilize the Cu(I) oxidation state.

In an embodiment, the step (e) reaction is performed at temperaturesbetween −20-300° C. for 0 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 5-120° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 20-100° C. for 0.5 to 18 h. In an embodiment, the step (c)reaction is performed at temperatures between 60-90° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 65-80° C. for 0.5 to 18 h.

In another embodiment, the invention provides a process for introducing2′-modifications into RNA, said process comprises a) obtaining RNA withan alkyne functional group at the 2′-position on at least one ribosering of an internal nucleotide; b) creating a solution of RNA in asolvent; c) adding an organic azide and a metal catalyst to the solutionto form a reaction and creating a 2′-modified RNA; and d) purifying the2¹-modified RNA.

In an embodiment, the step (a) RNA may be purchased or synthesized. Inan embodiment, the step (c) solvent is selected from aqueous buffersolutions (including phosphate buffers), aqueous DMSO, CH₃CN, DMF, DMAc,NMP and a suitable ionic liquid.

In an embodiment, the step (c) solvent is aqueous DMSO.

In an embodiment, the step (c) metal catalyst is selected from copperand ruthenium.

In an embodiment, the step (c) metal catalyst is copper.

In an embodiment, the step (c) metal catalyst is copper with a suitableligand to stabilize Cu(I) oxidation state.

In an embodiment, the step (c) reaction is performed at temperaturesbetween −20-300° C. for 0 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 5-120° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 20-100° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 60-90° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 65-80° C. for 0.5 to 18 h.

In an embodiment, the step (d) purification is performed inhigh-throughput format on 96-well C18 cartridges (solid-phaseextraction) or strong-anion-exchange-HPLC or reverse-phase HPLC orpoly(acrylamide) gel electrophoresis (PAGE) or size-exclusionchromatography.

In another embodiment, the invention provides a process for introducing2′-modifications into RNA, said process comprises a) obtaining RNA withan alkyne functional group at the 2′-position on at least one ribosering of an internal nucleotide; b) creating a solution of RNA in asolvent; c) adding an organic azide and a metal catalyst to the solutionto form a reaction and creating a 2′-modified RNA; d) cooling thesolution and adding a fluoride source; e) heating the solution; f)cooling the solution and adding a diluent; and g) purifying the2′-modified RNA.

In an embodiment, the step (a) RNA may be purchased or synthesized.

In an embodiment, the step (c) solvent is selected from aqueous buffersolutions (including phosphate buffers), aqueous DMSO, CH₃CN, DMF, DMAc,NMP and a suitable ionic liquid.

In an embodiment, the step (c) solvent is aqueous DMSO.

In an embodiment, the step (e) metal catalyst is selected from copperand ruthenium.

In an embodiment, the step (c) metal catalyst is copper.

In an embodiment, the step (c) metal catalyst is copper with a suitableligand to stabilize Cu(I) oxidation state.

In an embodiment, the step (c) reaction is performed at temperaturesbetween −20-300° C. for 0 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 5-120° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 20-100° C. for 0.5 to 18 h.

In an embodiment, the step (c) reaction is performed at temperaturesbetween 60-90° C. for 0.5 to 18 h. In an embodiment, the step (c)reaction is performed at temperatures between 65-80° C. for 0.5 to 18 h.

In an embodiment, the step (e) fluoride source is Et₃N.3HF,tetrabutylammonium fluoride, potassium fluoride and ammonium fluoride,

In an embodiment, the step (e) fluoride source is ammonium fluoride.

In an embodiment, the step (f) diluent is NaCl.

In an embodiment, the step (g) purification is performed inhigh-throughput format on 96-well C18 cartridges (solid-phaseextraction) or strong-anion-exchange-HPLC or reverse-phase HPLC orpoly(acrylamide) gel electrophoresis (PAGE) or size-exclusionchromatography.

In another embodiment, the instant invention also discloses a method forattaching targeting ligands to RNA utilizing the process describedherein.

In another embodiment, the instant invention further discloses a methodfor attaching targeting ligands to internal nucleotides in RNA utilizingthe process described herein.

In an embodiment, the invention provides a RNA with an alkyne functionalgroup at the 2′-position on one or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on two or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on three or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on four or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on five or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on six or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on seven or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on eight or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on nine or more ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on ten or more ribose rings.

In an embodiment, the invention provides a RNA with an alkyne functionalgroup at the 2′-position on one or more ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on two or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on three or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on four or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on five or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on six or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on seven or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on eight or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on nine or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a RNA with an alkynefunctional group at the 2′-position on ten or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In an embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on one or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on two or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on three or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on four or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on five or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on six or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on seven or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on eight or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on nine or more ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on ten or more ribose rings.

In an embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on one or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on two or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on three or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on four or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on five or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on six or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on seven or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on eight or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on nine or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a miRNA with an alkynefunctional group at the 2′-position on ten or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In an embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on one or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on two or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on three or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on four or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on five or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on six or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on seven or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on eight or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on nine or more ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on ten or more ribose rings.

In an embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on one or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on two or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on three or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on four or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on five or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on six or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on seven or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on eight or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on nine or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on ten or more ribose ringsexcluding the external 5′ and 3′ ribose rings.

In an embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on one ribose ring.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on two ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on three ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2¹-position on four ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on five ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on six ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on seven ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on eight ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on nine ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on ten ribose rings.

In an embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on one ribose ring excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on two ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on three ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on four ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on five ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on six ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on seven ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on eight ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on nine ribose rings excluding theexternal 5′ and 3′ ribose rings.

In another embodiment, the invention provides a siRNA with an alkynefunctional group at the 2′-position on ten ribose rings excluding theexternal 5′ and 3′ ribose rings.

In an embodiment, the invention provides the guide strand of the siRNAwith an alkyne functional group at the 2′-position on one ribose ring.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on two riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on three riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on four riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on five riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on six riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on seven riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on eight riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on nine riboserings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on ten riboserings.

In an embodiment, the invention provides the guide strand of the siRNAwith an alkyne functional group at the 2¹-position on one ribose ringexcluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on two riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on three riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on four riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on five riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on six riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on seven riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on eight riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on nine riboserings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the guide strand of thesiRNA with an alkyne functional group at the 2′-position on ten riboserings excluding the external 5′ and 3′ ribose rings.

In an embodiment, the invention provides the passenger strand of thesiRNA with an alkyne functional group at the 2′-position on one ribosering.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on tworibose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on threeribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on fourribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on fiveribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on sixribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on sevenribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on eightribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on nineribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on tenribose rings.

In an embodiment, the invention provides the passenger strand of thesiRNA with an alkyne functional group at the 2′-position on one ribosering excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on tworibose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on threeribose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on fourribose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on fiveribose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on sixribose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on sevenribose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on eightribose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on nineribose rings excluding the external 5′ and 3′ ribose rings.

In another embodiment, the invention provides the passenger strand ofthe siRNA with an alkyne functional group at the 2′-position on tenribose rings excluding the external 5′ and 3′ ribose rings.

Definitions “2′-modified RNA” means a RNA wherein at least one ribosering is modified at the 2′-position.

“Alkyne functional group” means any chemical compound containing analkyne functional group. The preferred “Alkyne functional group” is thepropargyl moiety shown throughout this disclosure.

“High-throughput format” means that several operations are run inparallel fashion such as for example in 96-well plate chemicalsynthesis, 96-well plate purification, 96-well plate chromatographicanalysis and 96-well plate mass spectrometric analysis.

“Internal nucleotide” means a nucleotide in an RNA molecule that is notat the 3′- or 5′-end. For example, the internal nucleotides in a 21 mersiRNA occur at positions 2-20.

“RNA” means a chemically modified or unmodified ribonucleic acidmolecule (single stranded or double stranded) comprising at least 3nucleotides, including but not limited to miRNA and siRNA. In anotherembodiment, “RNA” means miRNA. In another embodiment, “RNA” means siRNA.Chemical modifications include, for example, modifications to the base,ribose ring (excluding modifications to the 2′-position), and phosphatebackbone. The base can be a canonical base (A, G, T and U) or a modifiedor universal base (including but not limited to inosine andnitroindole).

“Organic azide” means any chemical compound containing the azidefunctional group.

“Metal catalyst” means any chemical form of copper and ruthenium,including solid-supported variants. Examples of metal catalyst includeCuBr, CuBrMe₂S, CuI, CuSO₄ or CuOAc and a suitable reducing agent suchas sodium ascorbate, Cu(CH₃CN)₄PF₆, CpRuCl(PPh₃)₂, and Cp*RuCl(PPh₃)₂.

“Ribose ring” means the ribose moiety in a ribonucleotide.

“Targeting ligand” means a conjugate delivery moiety capable ofdelivering an oligonucleotide to a target cell of interest. Targetingligands include, but are not limited to, lipids (cholesterol), sugars(NAG), proteins (transferrin), peptides, poly(ethylene)glycols andantibodies. See Juliano et al., Nucleic Acids Research, 2008, 1-14,doi:10.1093/narigkn342.

Utility

The present invention provides a process for introducing chemicalmodifications into RNA at the 2′-position on the ribose ring. It is wellknown in the art that RNA are useful for therapeutic and researchpurposes.

RNA Synthesis

The synthesis of RNA is well known in the art.

General Working Example “Click Reaction”

A suitable 2′-O-propargyl nucleoside phosphoramidite is incorporatedinto RNA using modern techniques based on the phosphoramidite approach.The crude, solid-support bound protected oligonucleotide is then treatedwith aqueous methylamine to remove nucleobase and phosphate protectinggroups. The crude product is then lyophilized to remove volatiles. Thecrude product is dissolved in DMSO:H₂O, treated with a suitable organicazide and a copper catalyst. After aging an appropriate amount of time,the reaction mixture is treated with fluoride to remove the2′-O-tert-butyldimethylsilyl protecting groups. The crude product isthen purified to obtain the chemically modified RNA.

Click Reaction between Benzyl Azide and RNA.

Lyophilized crude RNA (˜50 nmol) containing at least one alkynefunctional group (shown below) in 96-well format was dissolved inDMSO:water (75:25, 40 jut). Benzyl azide (1M in DMSO, 40 pi) was added,followed by a freshly prepared solution of CuBr.Me₂S in DMSO (12 mM, 40μL). The reaction block was sealed and heated at 65-80° C. overnight.The solution was cooled to room temperature and ammonium fluoride (100μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h,cooled to room temperature and diluted with 1M aqueous NaCl (800 μL).The crude product was purified on a C18 cartridge to afford the desiredchemically modified benzyl-triazole-linked RNA as determined by HPLC andLC-MS analyses.

Click Reaction between Azidomethyl Phenyl Sulfide and RNA.

Crude RNA (˜50 nmol) containing at least one alkyne functional group(shown below) was dissolved in DMSO:water (75:25, 40 μL). Azidomethylphenyl sulfide (1M in DMSO, 40 μL) was added, followed by a freshlyprepared solution of CuBr.Me₂S in DMSO (12 mM, 40 μL). The reactionblock was sealed and heated to 65-80° C. overnight. The solution wascooled to room temperature and ammonium fluoride (100 μL, 5.4M in water)was added. The solution was heated at 65° C. for 1 h, cooled to roomtemperature and diluted with 1M aqueous NaCl (800 μL). The crude productwas purified on a C18 cartridge to afford the desired chemicallymodified phenylthiomethyl-triazole-linked RNA as determined by HPLC andLC-MS analyses.

Click Reaction between Ethyl Azidoacetate and RNA.

Crude RNA (˜50 nmol) containing at least one alkyne functional group(shown below) was dissolved in DMSO:water (75:25, 40 μL). Ethylazidoacetate (1M in DMSO, 40 μL) was added, followed by a freshlyprepared solution of CuBr.Me₂S in DMSO (12 mM, 40 μL). The reactionblock was sealed and heated to 65-80° C. overnight. The solution wascooled to room temperature and ammonium fluoride (100 μL, 5.4M in water)was added. The solution was heated at 65° C. for 1 h, cooled to roomtemperature and diluted with 1M aqueous NaCl (800 μL). The crude productwas purified on a C18 cartridge to afford the desired chemicallymodified ethyl-carboxymethyl-1,4-triazole-linked RNA as determined byHPLC and LC-MS analyses.

Click Reaction between N-acetylgalactosamine Azide and RNA.

Crude RNA (˜50 nmol) containing at least one alkyne functional group(shown below) was dissolved in DMSO:water (75:25, 40 μL). ModifiedN-acetyl galactosamine azide (1M in DMSO, 40 μL) was added, followed bya freshly prepared solution of CuBr.Me₂S in DMSO (12 mM, 40 μL). Thereaction block was sealed and heated to 65-80° C. overnight. Thesolution was cooled to room temperature and ammonium fluoride (100 μL,5.4M in water) was added. The solution was heated at 65° C. for 1 h,cooled to room temperature and diluted with 1M aqueous NaCl (800 μL).The crude product was purified on a C18 cartridge to afford the desiredchemically modified N-acetylgalactosamine-1,4-triazole-linked RNA asdetermined by HPLC and LC-MS analyses.

Click Reaction between N-acetylgalactosamine Azide and RNA(Multi-Click).

Crude RNA (˜50 nmol) containing more than one alkyne functional group(shown below) was dissolved in DMSO:water (75:25, 40 μL). ModifiedN-acetylgalactosamine azide (1M in DMSO, 40 μL) was added, followed by afreshly prepared solution of CuBr.Me₂S in DMSO (12 mM, 40 μL). Thereaction block was sealed and heated to 65-80° C. overnight. Thesolution was cooled to room temperature and ammonium fluoride (100 μL,5.4M in water) was added. The solution was heated at 65° C. for 1 h,cooled to room temperature and diluted with 1M aqueous NaCl (800 μL).The crude product was purified on a C18 cartridge to afford the desiredchemically modified N-acetylgalactosamine-1,4-triazole-linked RNA asdetermined by HPLC and LC-MS analyses.

Click Reaction Walkthrough between Benzyl Azide and SSB(291) RNA.

Crude RNA (50 nmol) containing at least one alkyne functional group(shown below) was dissolved in DMSO:water (75:25, 40 uL). Benzyl azide(1M in DMSO, 40 uL) was added, followed by a freshly prepared solutionof CuBr.Me₂S in DMSO (12 mM, 40 uL). The reaction block was sealed andheated at 65-SO ° C. overnight. The solution was cooled to roomtemperature and ammonium fluoride (100 μL, 5.4M in water) was added. Thesolution was heated at 65° C. for 1 h, cooled to room temperature anddiluted with 1M aqueous NaCl (800 uL). The crude product was purified ona C18 cartridge to afford the desired chemically modifiedbenzyl-1,4-triazole-linked RNA as determined by HPLC and LC-MS analyses.

Click Reaction between 11-azido-3,6,9-trioxaundecan-1-amine and SSB(291)RNA.

Crude RNA (50 nmol) containing at least one alkyne functional group(shown below) was dissolved in DMSO:water (75:25, 40 uL).11-Azido-3,6,9-trioxaundecan-1-amine (1M in DMSO, 40 uL) was added,followed by a freshly prepared solution of CuBr.Me₂S in DMSO (12 mM, 40uL). The reaction block was sealed and heated at 65-80° C. overnight.The solution was cooled to room temperature and ammonium fluoride (100μL, 5.4M in water) was added. The solution was heated at 65° C. for 1 h,cooled to room temperature and diluted with 1M aqueous NaCl (800 uL).The crude product was purified on a C18 cartridge to afford the desiredchemically modified amino-PEG-1,4-triazole-linked RNA as determined byHPLC and LC-MS analyses.

Click Reaction on Unprotected “Free” RNA.

Purified deprotected free RNA (8.6 mg, sequence=UUA CAU UAA(2′-propargylabasic)GU CUG UUG UdTdT) was dissolved in DMSO:water{75:25, 1 mL). The solution (75 μL) was dispensed in wells containingstir bars. A bright blue-green solution (75 μL) oftris(1-(O-ethylcarboxymethyl)-1 H-1,2,3-triazol-4-ylmethyl)amine ligand(50 mg) and CuBr (10 mg, 99.999%) in DMSO:water (75:25, 5 mL) was added.Phenylthiomethyl azide (5 μL) was added. The reaction block was sealedand agitated overnight at room temperature. The crude product waspurified to afford the desired chemically modifiedphenylthiomethyl-1,4-triazole-linked RNA as determined by HPLC and LC-MSanalyses.

Click Reaction on Unprotected “Free” RNA

Purified deprotected free RNA (8.6 mg, sequence=UUA CAU UAA(2′-propargylabasic)GU CUG UUG UdTdT) was dissolved in DMSO:water(75:25, 1 mL). The solution (75 μL) was dispensed in wells containingstir bars. A bright blue-green solution (75 μL) oftris(1-(O-ethylcarboxymethyl)-1H-1,2,3-triazol-4-ylmethyl)amine ligand(50 mg) and CuBr (10 mg, 99.999%) in DMSO:water (75:25, 5 mL) was added.Benzyl azide (5 μL) was added. The reaction block was sealed andagitated overnight at room temperature. The crude product was purifiedto afford the desired chemically modified benzyl-1,4-triazole-linked RNAas determined by HPLC and LC-MS analyses.

Click reaction on Unprotected “Free” RNA

Purified deprotected free RNA (8.6 mg, sequence=UUA CAU UAA(2′-propargylabasic)GU CUG UUG UdTdT) was dissolved in DMSO:water(75:25, 1 mL). The solution (75 μL) was dispensed in wells containingstir bars. A bright blue-green solution (75 μL) oftris(1-(O-ethylcarboxymethyl)-1H-1,2,3-triazol-4-ylmethyl)amine ligand(50 mg) and CuBr (10 mg, 99.999%) in DMSO:water (75:25, 5 mL) was added.Ethyl azidoacetate (15 μL, 25-30% wt in EtOH) was added. The reactionblock was sealed and agitated overnight at room temperature. The crudeproduct was purified to afford the desired chemically modified ethylcarboxymethyl-1,4-triazole-linked RNA as determined by HPLC and LC-MSanalyses.

Assays

Position in Guide strand Gene mRNA sequence sequence (5′-3′) SSB 291UUACAUUAAAGUCUGUUGU Luc 80 UAUCUCUUCAUAGCCUUAU

Positions 1-19 of both strands were ribonucleotides, and the overhangsat positions 20 and 21 contained 2′-deoxyribonucleotide thymidines. Thisunmodified siRNA was the template for systematic evaluation of modifiedsiRNAs containing a single modification at every position along theguide strand. In order to examine the effect of the chemicalmodifications for the SSB sequence, we synthesized the RNA oligomerswith the first nucleotide, uridine (U), replaced with2′-O-propargyl-inosine. Then, a second sequence, in which the secondnucleoside (U) was replaced with 2′-O-propargyl-inosine was synthesized,keeping all other nucleotides unchanged. Altogether nineteen sequenceswere synthesized where the universal base replaced all the naturalnucleosides in that sequence. This “modification walkthrough” isdepicted in Table 1 for SSB(291). The desired chemical modification wasthen introduced into the assembled RNA by the methods described inSchemes 6 and 7.

TABLE 1 Position in Guide strand Entry Gene mRNA sequencesequence (5′-3′) unmodified SSB 291 UUACAUUAAAGUCUGUUGU 1 SSB 291XUACAUUAAAGUCUGUUGU 2 SSB 291 UXAXAUUAAAGUCUGUUGU 3 SSB 291UUXCXUUAAAGUCUGUUGU 4 SSB 291 UUAXAUUAAAGUCUGUUGU 5 SSB 291UUACXUUAAAGUCUGUUGU 6 SSB 291 UUACAXUAAAGUCUGUUGU 7 SSB 291UUACAUXAAAGUCUGUUGU 8 SSB 291 UUACAUUXAAGUCUGUUGU 9 SSB 291UUACAUUAXAGUCUGUUGU 10 SSB 291 UUACAUUAAXGUCUGUUGU 11 SSB 291UUACAUUAAAXUCUGUUGU 12 SSB 291 UUACAUUAAAGXCUGUUGU 13 SSB 291UUACAUUAAAGUXUGUUGU 14 SSB 291 UUACAUUAAAGUCXGUUGU 15 SSB 291UUACAUUAAAGUCUXUUGU 16 SSB 291 UUACAUUAAAGUCUGXUGU 17 SSB 291UUACAUUAAAGUCUGUXGU 18 SSB 291 UUACAUUAAAGUCUGUUXU 19 SSB 291UUACAUUAAAGUCUGUUGX (X represents a universal base such as inosine) SSBKnockdown

In a 96-well format, Hepa1-6 cells were transfected with 10 nM of eitherthe unmodified, modified, or negative control siRNA using a commerciallipid transfection reagent. The target mRNA was assessed for degradationusing standard Taqman procedures.

Modified Multiplex Luciferase Report Assay for In Vitro Duration StudyAssay Principle:

Multiplex luciferase assay for in vitro duration study is modified fromthe manufacturer's instruction using HeLa-luc cell line. Briefly, thecell viability and the luciferease expression at the same well aredetermined by CellTiter-Fluor™ (Promega, Cat #G6082) and Bright-Glo™(Promega Cat #E2620) sequentially.

HeLa-luc cell line is a stable firefly luciferase reporter expressioncell line. Bright-Glo™ luciferase assay system contains the stablesubstrate—luciferin and assay buffer. The luminescent reaction ofluciferease and luciferin has high quantum yield and can be detected asluminescence intensity, which represents the luciferase expressionlevel.

Target siRNAs containing luciferase coding region is designed to betransfected into the HeLa-luc cells. Once the target is effected, theluciferase expression is reduced accordingly, Therefore, the siRNAsilencing efficacy can be determined by the relative luminecenceintensity of treated cells.

To reduce the variation caused by cell viability and cell platingprocess, the cell viability of the same treatment wells is measuredusing CellTiter-fluor kit. This assay measures the conserved andconstitutive protease activity within live cells and therefore serves asa marker of cell viability, using a fluorogenic, cell-permeable peptidesubstrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC).

Experiment Design:

Luciferase stable expressed HeLa-luc cell cells are plated in 96-wellplates at density of 4,500 cells per well in 100 μL DMEM media withoutantibiotics 24 hours prior to transfection. siRNA transfection isperformed using the RNAiMAX™ (Invitrogen). Briefly, 0.05 μM siRNA aremixed with Opti-MEMmedia and RNAiMAX and incubated at room temperaturefor 15 min. The mix is then added to the cells. The final siRNAconcentration is 1 nM. Cell plates for all time points are transfectedat same time with a medium change at 6 hours post-transfection into 100μL of fresh completed DMEM (DMEM+10% FBS+Pen/strep).

In vitro duration is determined by the luciferase expressionpost-transfection at four time points: day 1, day 2, day 5 and day 7.Addition medium changes are performed at day 2 and day 5 into 100 μL offresh completed DMEM (DMEM+10% FBS+Penn/strep). Luciferase levels aredetermined using the Bright-Glo Luminescence Assay (Promega) andmeasuring the wells on an Envison instrument (Perkin Elmer) according tomanufacturer's instructions.

To reduce the variation caused by cell viability and cell platingprocess, the cell viability of the same treatment wells is measuredusing CellTiter-fluor kit (Promega) according to manufacturer'sinstructions. This assay measures the conserved and constitutiveprotease activity within live cells and therefore servers as a marker ofcell viability, using a fluorogenic, cell-permeable peptide substrate(glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC). The fluorescence wasmeasured on the Envision using exciton filter at 405 nm and emissionfilter at 510 nm.

The luciferase expression was normalized to cell viability. The log ofthis number was calculated to determine the luciferase protein that wasdegraded (knockdown). A non-targeting siRNA was subtracted from thisvalue to account for non-specific background.

EXAMPLES

The following Examples 1-6 were generated utilizing the Assays above anddemonstrate the utility of the RNAs made by the methods described in theSchemes. As demonstrated, the RNAs made by the process of the inventionare useful in high-throughput structure-activity relationship studies onchemically modified RNA in 96-well format.

Example 1

In FIG. 1, the impact on knockdown of the 2′-O-benzyl-triazole inosinechemical modification was systematically evaluated along positions 1through 19 of the guide strand of an siRNA targeting mRNA SSB(291).

Example 2

In FIG. 2, the impact of the 2′-O-phenylthiomethyl-triazole inosinechemical modification was systematically evaluated along positions 1through 19 of the guide strand of an siRNA targeting mRNA SSB(291).

Example 3

In FIG. 3, the impact on knockdown of the 2′-O-benzyl-triazole inosinechemical modification was systematically evaluated along positions 1through 19 of the guide strand of an siRNA targeting mRNA Luc(80).

Example 4

In FIG. 4, the impact of the 2i-O-phenylthiomethyl-triazole inosinechemical modifications were systematically evaluated along positions 1through 19 of the guide strand of an siRNA targeting mRNA Luc(80).

Example 5

In FIG. 5, the impact on duration of knockdown activity of the2′-O-benzyl-triazole inosine chemical modification was systematicallyevaluated along positions 1 through 19 of the guide strand of an siRNAtargeting mRNA Luc(80).

Example 6

In FIG. 6, the impact on duration of knockdown activity of the2′-O-phenylthiomethyl inosine chemical modification was systematicallyevaluated along positions 1 through 19 of the guide strand of an siRNAtargeting mRNA Luc(80).

Example 7

In FIG. 7, the liver targeting compound N-acetyl-galactosamine (NAG) canbe introduced as a chemical modification that may help with specificcell targeting, cellular uptake and delivery of RNA.

Example 8

In FIG. 8, poly(ethylene)glycol amines can be introduced to improvesolubility properties, cellular uptake, immune stealth, reduce metabolicclearance and delivery of RNA.

Example 9

In FIG. 9, the “click” reaction can be utilized to introduce multiplechemical modifications in one synthetic operation. For example, theclick reaction was performed to introduce three units of protectedN-acetylgalactosamine on RNA. This may lead to improved physicalproperties towards solubility, cellular uptake, and delivery of siRNA.

1. A process for introducing 2′-modifications into RNA, said processcomprises a) obtaining RNA with an alkyne functional group at the2′-position on at least one ribose ring; b) creating a solution of RNAin a solvent; and c) adding an organic azide and a metal catalyst to thesolution to form a reaction and creating a 2′-modified RNA.
 2. Theprocess of claim 1, wherein the process is conducted in high-throughputformat.
 3. The process of claim 1, wherein the solvent of step (b) isselected from the group consisting of an aqueous buffer solution,aqueous DMSO, CH₃CN, DMF, DMAc, NMP and a suitable ionic liquid.
 4. Theprocess of claim 1, wherein the metal catalyst of step (c) is selectedfrom copper and ruthenium.
 5. The process of claim 4, wherein the metalcatalyst of step (c) is Cu(I) and a suitable ligand to stabilize theCu(I) oxidation state.
 6. The process of claim 1, wherein step (c) isperformed at a temperature of between −20° C. to 300° C.
 7. The processof claim 6, wherein step (c) is performed at a temperature of between20° C. to 100° C. for 0.5 to 18 hours.
 8. The process of claim 1,wherein the RNA in step a) has an alkyne functional group at the2′-position on at least one ribose ring of an internal nucleotide.
 9. Aprocess for introducing 2′-modifications into RNA, said processcomprises a) obtaining RNA with an alkyne functional group at the2′-position on at least one ribose ring of an internal nucleotide; b)creating a solution of RNA in a solvent; c) adding an organic azide anda metal catalyst to the solution to form a reaction and creating a2′-modified RNA; and d) purifying the 2′-modified RNA.
 10. A process forintroducing 2′-modifications into RNA, said process comprises a)obtaining RNA with an alkyne functional group at the 2′-position on atleast one ribose ring of an internal nucleotide; b) creating a solutionof RNA in a solvent; c) adding an organic azide and a metal catalyst tothe solution to form a reaction and creating a 2′-modified RNA; d)cooling the solution and adding a fluoride source; e) heating thesolution; f) cooling the solution and adding a diluent; and g) purifyingthe 2′-modified RNA.